Botulinum neurotoxins (BoNTs) are classified as one of the six highest-risk threat agents for bioterrorism (the 'Category A agents'). BoNTs have been produced and weaponized by rogue nations and deployed by terrorist groups. The overall aim of this application is to generate neutralizing human compatible monoclonal antibodies (mAbs) to the BoNTs for prevention and treatment of botulism resulting from intentional exposure to toxin. Achievement of this goal requires three components: 1) determination of the extent of BoNT gene diversity of the seven toxin serotypes (A-G); 2) generation of panels of broadly neutralizing human compatible mAbs to the seven BoNT serotypes; and 3) in vitro and in vivo characterization of mAbs with respect to serotype cross reactivity and neutralization. These goals will be achieved by an inter-institutional team of botulism experts who have worked in this field for more than ten years: Eric Johnson, BoNT genetics and toxin production; James D. Marks, antibody engineering; and Leonard Smith, BoNT vaccine development and animal models of toxin neutralization. They will be assisted by one of the pioneers of microbial genetic characterization, Paul J. Jackson. The extent of toxin genetic diversity will be determined by establishment of a large repository of geographically disperse Clostridial strains that produce BoNTs. Strains will be characterized by pulsed field gel electrophoresis and amplified fragment length polymorphism (AFLP). These results will guide sequencing of selected BoNT genes to determine the extent of toxin diversity within and between serotypes. Based on these results, BoNT's and recombinant BoNT fragments will be expressed and purified and used as immunogens for mAb generation. Phage antibody libraries will be constructed from the V-genes of immunized mice, humans, and mice transgenic for the human irnmunoglobulin locus. High affinity, potently neutralizing human compatible (human or humanized) antibodies to each of the BoNT serotypes will be selected from these libraries. Where necessary, murine antibodies will be humanized and mAb affinity increased by molecular evolution to achieve potent toxin neutralization. It is anticipated that at the end of the five year project period, a panel of mAbs will have been generated which in the relevant animal models broadly neutralize each of the seven BoNT serotypes. All antibodies will be human compatible (either humanized or fully human in sequence) and will be ready for transfer to a manufacturing facility for cGMP production, toxicology studies, and human testing. It is also anticipated that the project will define the range of BoNT diversity and determine how this diversity affects immunogenicity and antibody recognition. Such information will be invaluable for vaccine development as well as diagnostic testing and microbial forensics.